Hematopoiesis involves a series of tightly regulated molecular events. The mechanisms controlling the decision of the multi-potent stem/progenitor cell to self-renew or differentiate are not well understood. Many initial developmental decisions are regulated by the homeobox family of transcription factors. The hematopoietically expressed homeobox gene, Hex, is an interesting candidate gene in the study of early hematopoietic differentiation. The goal of the proposed studies is to enhance our current understanding of the role Hex play in hematopoietic lineage commitment. The role Hex plays in embryonic stem (ES) cell differentiation into the hematopoietic progenitor will be examined by three specific aims: 1) directed knockdown of Hex gene expression in order to elucidate whether Hex is necessary from hematopoietic commitment. 2) overexpression of Hex to determine whether Hex is sufficient to induce hematopoietic commitment. 3) microarray analysis to determine the downstream targets of Hex which mediate hematopoietic differentiation. ES cells provide an ideal model system for these studies. In vitro, they recapitulate the development of the early hematopoietic lineages and all myeloid lineages. To model hematopoiesis, an in vitro murine ES cell co-culture system was adapted from Era and Witte. Under normal conditions, undifferentiated ES cells are maintained in LIF. When cultured on OP-9 stromal cells, ES cells differentiate into hemangioblasts by day 5. Continued co-culture with OP-9 cells results in the differentiation of ES cells into hematopoietic precursors (day 8) and mature, terminally differentiated lineages by day 14. Hematopoietic cells can then be analyzed for cell surface markers by flow cytometry and morphologically by cytoprep. The use of a Tetracyline- inducible system (Tet-Off), within the ES cells, allows one to tightly regulate the timing and amount of Hex expression. The goal of the proposed experiments is to characterize the effects of Hex transcriptional activity and Hex gene knockdown on hematopoietic cell survival, apoptosis, proliferation and differentiation. Overall these experiments are designed to more clearly elucidate the molecular pathways that regulate hematopoietic stem cell differentiation. This work will provide important insights relevant to a number of blood cell disorders including cancer, anemias and immune disorders.